MEDyC Unité CNRS UMR6237, UFR de Pharmacie, IFR53, 51096 Reims, France.
Oncol Rep. 2012 Jan;27(1):232-7. doi: 10.3892/or.2011.1468. Epub 2011 Sep 20.
Resistance to etoposide has been associated with the overexpression of P-glycoprotein and MRP1 in human tumor cells. However, the role of BCRP in resistance to etoposide has not been clearly established, especially the significance of arginine 482 mutations in drug transport (cellular uptake and efflux). Different levels of resistance to etoposide have been recently observed in cells expressing BCRP in terms of cytotoxicity. The aim of this work was to study the effects of these mutations on the functional involvement of BCRP in etoposide transport. HEK293 cells were transfected with an empty vector (HEK/V), the vector bearing the wild-type BCRP (HEK/R482), the mutant arginine-482-glycine (HEK/R482G) or the mutant arginine-482-threonine (HEK/R482T). MTT assay was used to study the cytotoxic effect of etoposide and [3H]-etoposide was used to determine cellular drug uptake and efflux. Data show that HEK/R482G cells displayed the highest levels of resistance to etoposide. Cellular [3H]-etoposide uptake was lower in HEK/R482, HEK/R482G and HEK/R482T cells compared to HEK/V cells. In addition, cellular [3H]-etoposide uptake in HEK/R482G was the lowest. Drug efflux measurements showed that fumitremorgin C was able to increase the residual cellular [3H]-etoposide uptake in BCRP-transfected cells and especially in HEK/R482G ones. Our data show that the R482G mutation in BCRP is able to increase efflux of etoposide and that mutation analysis at codon 482 may be of clinical importance in cancers treated with etoposide.
对依托泊苷的耐药性与人类肿瘤细胞中 P-糖蛋白和 MRP1 的过度表达有关。然而，BCRP 在依托泊苷耐药中的作用尚未明确确定，特别是在药物转运（细胞摄取和外排）中精氨酸 482 突变的意义。最近在表达 BCRP 的细胞中观察到，在细胞毒性方面对依托泊苷的耐药性存在不同水平。这项工作的目的是研究这些突变对 BCRP 参与依托泊苷转运的功能的影响。用空载体（HEK/V）、携带野生型 BCRP 的载体（HEK/R482）、突变精氨酸-482-甘氨酸（HEK/R482G）或突变精氨酸-482-苏氨酸（HEK/R482T）转染 HEK293 细胞。MTT 测定用于研究依托泊苷的细胞毒性作用，并用 [3H]-依托泊苷测定细胞内药物摄取和外排。数据表明，HEK/R482G 细胞对依托泊苷的耐药性最高。与 HEK/V 细胞相比，HEK/R482、HEK/R482G 和 HEK/R482T 细胞中的细胞内 [3H]-依托泊苷摄取较低。此外，HEK/R482G 细胞中的细胞内 [3H]-依托泊苷摄取最低。药物外排测量表明， fumitremorgin C 能够增加 BCRP 转染细胞，特别是 HEK/R482G 细胞中的残留细胞内 [3H]-依托泊苷摄取。我们的数据表明，BCRP 中的 R482G 突变能够增加依托泊苷的外排，并且在接受依托泊苷治疗的癌症中，密码子 482 处的突变分析可能具有临床意义。
